Summary: The purpose of this project is to identify and characterize virulence determinants in Corynebacterium diphtheriae in order to understand how this important pathogen causes disease. Expression of diphtheria toxin, the primary virulence determinant of C. diphtheriae, is regulated by iron, and it is likely that additional virulence factors are coordinately regulated with that of the toxin. The ability to acquire iron during an infection is essential for many bacterial pathogens to cause disease. It was shown in earlier studies from this laboratory that the hmuO gene is required for the utilization of iron from heme and hemoglobin in C. diphtheriae. Recent studies have focused on; 1) understanding the molecular mechanisms of hmuO gene regulation, 2) the characterization of factors involved the transport of heme in C. diphtheriae and 3) the development of genetic tools in C. diphtheriae to more accurately define the function of various genetic systems. Transcription from the hmuO promoter has been shown to be under dual regulation in which expression is positively regulated by a heme source, such as heme or hemoglobin, and negatively regulated by the diphtheria toxin repressor protein (DtxR) and iron. Studies in this laboratory have shown that heme-dependent activation of the hmuO promoter occurs through a two component signal transduction system that requires the chrA and chrS genes, which encode a response regulator and sensor kinase, respectively. An ABC (ATP-binding cassette) type transport system is involved in the utilization of heme and hemoglobin as iron sources in C. diphtheriae and the related pathogen C. ulcerans. The three proteins identified in this system are homologous to proteins involved in heme transport from gram-negative bacteria. The HmuT protein, which is a lipoprotein, is proposed to be the cell surface receptor for heme. A system for constructing defined mutation was developed for both C. diphtheriae and C. ulcerans. Mutations in the gene encoding the heme receptor, hmuT, were constructed in both Corynebacterium species. Disruption of the hmuT gene in C. ulcerans resulted in an inability to use heme as an iron source, while inactivation of hmuT in C. diphtheriae had no effect on heme transport, suggesting this species has an additional mechanism for transporting heme. The heme transport locus from C. ulcerans was also cloned and sequenced and was shown to be approximately 70-80 homologous to the heme transport locus from C. diphtheriae.